This invention relates to a method for the purification of lipoproteins in order to provide a material high in cholesterol content which is suitable for use as a cholesterol reference standard for determining the cholesterol content of body fluids.
The analytical determination of serum cholesterol levels is a useful tool in screening for coronary artery disease, diabetes mellitus, nephrosis, hepatic and thyroid diseases as well as metabolic disorders caused by endocrine disturbances. Clinical chemistry procedures designed to facilitate the determination of cholesterol levels in serum, generally involve the use of a cholesterol control reference standard which comprises human serum and certain organic salts as components. In U.S. Pat. No. 4,290,774 there is described the desirability of a cholesterol reference material which is not used in conjunction with human serum or plasma and points out the difficulties inherent in recovering complexes formed by adsorbing lipoproteins to adsorbents to form adsorbent/lipoprotein complexes. This patent describes such a procedure which involves adsorbing a lipoprotein containing substance onto a silica adsorbent, separating the absorbed lipoprotein from the excess solution, freezing and thawing the adsorbed lipoprotein, eluting the absorbed lipoprotein at a pH of from 10 to 11.5, concentrating the lipoprotein to a desired concentration, adjusting the pH to a level of from 7.0 to 10.0; adjusting the salt concentration to a level of less than 0.05M, heating the resultant to a temperature of from 50.degree. to 100.degree. C., adding an alkaline carbonate and alkaline earth salt to form a precipitate, removing the precipitate, adjusting the pH to a level of from 6.5 to 9.0 and recovering the purified cholesterol. The specific silica adsorbent described in this patent is Cabosil.RTM. ultrafine silica obtainable from Cabot Corporation of Boston, Mass.
An improvement to the procedure disclosed in the '774 patent is disclosed in U.S. Pat. No. 4,762,792 which improvement involves eluting the silica adsorbed lipoprotein at a pH of from about 10 to 11.5, adjusting the salt concentration to less than about 0.05M, heating the eluted lipoprotein to a temperature of from about 50.degree. to 100.degree. C. for a time sufficient to increase its storage stability, whereupon the lipoprotein fraction is dialyzed against an alkaline material such as aqueous sodium carbonate, adjusting the pH from about 6.5 to 9.0 and recovering the lipoprotein cholesterol. This patent states that the silica adsorbent does not have a critical composition and describes the Cabosil.RTM. product as well as powdered silica available under the tradename Aerosil.RTM. 380 from the Cary Company as being suitable.
While these prior art procedures work well for the recovery of cholesterol from serum or plasma, they suffer from the disadvantage that the liquid-solid separation is, as a practical matter, limited to centrifugation; attempts at filtration have been found to be minimally successful and labor intensive. Furthermore, in order to effectively remove solubilized Aerosil.RTM. silicates from the high pH eluate, a freeze-thaw of the eluate is required. The freeze-thaw procedures causes significant silicate precipitation. While the precipitate may be removed by centrifugation, this process is time consuming and labor intensive.
Amorphous silica, i.e. that form of SiO.sub.2 which lacks a crystal structure, has been used as an adsorbent since at least as early as World War I when it was considered for use as an adsorbent in gas masks. The microparticulate silicas include pyrogenic silicas (also known as fumed silica) and silicas precipitated from aqueous solution. In the preparation of fumed silica such as the previously mentioned, Cabosil.RTM. and Aerosil.RTM. products, sand is vaporized at about 2000.degree. C. On cooling, anhydrous amorphous silica powders form in the presence of a reducing agent such as coke. The amorphous silica sublimes at about 1500.degree. C. to provide Si which is then oxidized to produce very finely divided particulate SiO.sub.2. Commercially available fumed silica will typically have an average particle size in the range of from about 7 to 16nm.
Precipitated silica (also called particulate silica) is composed of aggregates of ultimate particles of colloidal size that have become linked in a gel network. Precipitated silicas, such as Sipernat.RTM. from Degussa, are typically formed by the precipitation of pyrogenic silica from solution.
U.S. Pat. No. 5,151,872 discloses the use of fumed silica for the adsorption of lipoproteins from plasma. The patentees point out that this procedure was known before their invention but disclose the improvement of selectively desorbing HDL from the fumed silica by incubating with a detergent containing formulation.
In commonly assigned allowed application, U.S. Ser. No. 990,592, there is disclosed a method for selectively separating high density lipoprotein from blood by contacting the blood with finely divided, porous silica. The porous silica is described comprising particles of from 1.mu. to 1000.mu. in their longest dimension which have surface pores of from about 80 .ANG. to 1000 .ANG. in size. This reference illustrates the use of Vydac porous silica of 4.mu. particle size having a pore size of 300 .ANG. in its Example I. Microporous silica gels are obtained by heating a hydrated gel at 1000.degree. C. for about 10 hours. The '592 application is concerned with the use of large pore silicas and silicates such as microporous silica, silica gel and controlled pore glass as selective adsorbent materials for HDL from blood serum or plasma.